PROJECT SUMMARY/ABSTRACT Antibiotic resistance is a major healthcare problem because it renders treatment ineffective, leading to chronic infections and death. Pseudomonas aeruginosa (PSA) is notorious for its capacity to mutate and become antibiotic resistant during chronic infections. Evolutionary theory predicts that these mutations will lead to fitness defects, including decreased growth rates that reduce the resistant organism?s ability to survive competition with wild-type susceptible siblings when antibiotic treatment ends. Moreover, fitness costs could also reduce virulence by reducing the resistant population?s ability to replicate fast enough to overcome killing by the host immune response. In reality, these predictions are frequently contradicted: antibiotic resistant PSA are readily isolated alongside susceptible siblings during decades-long chronic infections, and recently we discovered that antibiotic resistance mutations that increase efflux pump expression can also increase PSA virulence even in the absence of antibiotics. However, the conditions that enable resistant bacteria to compete with susceptible bacteria are unknown and the mechanisms by which resistance mutations increase virulence are unclear. The central hypothesis of this proposal is that mutation of efflux pump components leads to increased virulence in antibiotic resistant strains. The three specific aims of this proposal are to 1) determine the role of quorum sensing in enhanced virulence of aztreonam resistant PSA, 2) determine fitness of aztreonam resistant mutants relative to wild-type PSA, and 3) identify novel therapeutic targets in aztreonam-resistant PSA. In our previous work, both hypervirulent resistant PSA strains had resistance mutations that functioned by increasing mexAB-oprM antibiotic efflux pump expression. In addition to its role exporting antibiotics, MexAB-OprM also secretes a PSA quorum sensing molecule which is involved in regulating several virulence factors. In preliminary experiments, hypervirulent resistant PSA increased swarming and biofilm phenotypes, which are both mediated by the bacterial quorum sensing regulated virulence factor rhamnolipid. In the first aim of this proposal we will test the hypothesis that increased virulence of resistant PSA is due to increased quorum sensing regulated rhamnolipid production. In the second aim, we will test the hypothesis that specific nutrients, stresses, or host cells enable resistant bacteria to compete with susceptible bacteria. In the third aim, we will test the hypothesis that targeted therapies against hypervirulent resistant PSA will reduce virulence and improve clearance during infection, and we will use random transposon mutagenesis in combination with next-generation sequencing to identify new drug targets in antibiotic resistant PSA. These studies will help us understand how antibiotic resistant bacteria can arise during infection, as well as how we can leverage this knowledge and translate it into desperately needed therapies for antibiotic resistant bacteria.